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1.
Nat Commun ; 10(1): 2110, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068593

RESUMO

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Ribossomos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral/transplante , Movimento Celular/fisiologia , Nucléolo Celular/metabolismo , Embrião de Galinha , Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Ribossômico/metabolismo , Ribossomos/genética
2.
J Exp Clin Cancer Res ; 38(1): 50, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717766

RESUMO

BACKGROUND: Altered glucose metabolism endows tumor cells with metabolic flexibility for biosynthesis requirements. Phosphoenolpyruvate carboxykinase 1 (PCK1), a key enzyme in the gluconeogenesis pathway, is downregulated in hepatocellular carcinoma (HCC) and predicts poor prognosis. Overexpression of PCK1 has been shown to suppress liver tumor growth, but the underlying mechanism remains unclear. METHODS: mRNA and protein expression patterns of PCK1, AMPK, pAMPK, and the CDK/Rb/E2F pathway were determined using qRT-PCR and western blotting. Cell proliferation ability and cell cycle were assessed by MTS assay and flow cytometric analysis. The effect of PCK1 on tumor growth was examined in xenograft implantation models. RESULTS: Both gain and loss-of-function experiments demonstrated that PCK1 deficiency promotes hepatoma cell proliferation through inactivation of AMPK, suppression of p27Kip1 expression, and stimulation of the CDK/Rb/E2F pathway, thereby accelerating cell cycle transition from the G1 to S phase under glucose-starved conditions. Overexpression of PCK1 reduced cellular ATP levels and enhanced AMPK phosphorylation and p27Kip1 expression but decreased Rb phosphorylation, leading to cell cycle arrest at G1. AMPK knockdown significantly reversed G1-phase arrest and growth inhibition of PCK1-expressing SK-Hep1 cells. In addition, the AMPK activator metformin remarkably suppressed the growth of PCK1-knockout PLC/PRF/5 cells and inhibited tumor growth in an orthotropic HCC mouse model. CONCLUSION: This study revealed that PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis and supports a potential therapeutic and protective effect of metformin on HCC.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Hepatocelular/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/patologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Regulação para Baixo , Fatores de Transcrição E2F/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Mol Med ; 43(2): 739-748, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569159

RESUMO

Biliary atresia (BA) is the most common cause of chronic cholestasis in children. The long non­coding RNA (lncRNA) Annexin A2 pseudogene 3 (ANXA2P3) and Annexin A2 (ANXA2) have been suggested to serve pivotal roles in BA; however, the clinical significance and biological roles of ANXA2P3 and ANXA2 in BA remain to be elucidated. The present study aimed to elucidate the function of ANAX2P3 and ANXA2 in BA­induced liver injury using a human liver cell line and liver tissues from patients with BA. Reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemistry were conducted to determine the expression levels of ANXA2 and ANXA2P3 in liver tissues from patients with BA. Classification of fibrosis was analyzed by Masson staining. The functional roles of ANXA2 and ANXA2P3 in liver cells were determined by Cell Counting kit­8 assay, and flow cytometric and cell cycle analyses. Activation of the ANXA2/ANXA2P3 signaling pathway in liver cells was evaluated by western blot analysis. According to the present results, the expression levels of ANXA2 and ANXA2P3 were significantly increased in liver tissues from patients with BA. In addition, knocking down the expression of ANXA2P3 and ANXA2 may result in reduced liver cell proliferation, cell cycle arrest in G1 phase and increased apoptosis of liver cells in vitro. Furthermore, in cells in which ANXA2 and ANXA2P3 were overexpressed, cell apoptosis was reduced and cell cycle arrest in G2 phase. Taken together, these results indicated that ANXA2P3 and ANXA2 may have protective effects against liver injury progression and may be considered biomarkers in patients with BA.


Assuntos
Anexina A2/fisiologia , Atresia Biliar/metabolismo , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Longo não Codificante/fisiologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Linhagem Celular , Pré-Escolar , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Lactente , Pseudogenes/fisiologia
4.
Int J Oncol ; 52(4): 1224-1234, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29532883

RESUMO

This study examined the effects of long non­coding RNA (lncRNA) BC032020 on the development of human pancreatic ductal adenocarcinoma (PDAC), and the potential molecular mechanisms responsible for these effects. The expression of BC032020 was assessed in 20 pairs of PDAC tumor tissues and adjacent normal tissues. The overexpression of BC032020 was enforced in the AsPC­1 and PANC­1 cells, and the effects on cell proliferation, cell cycle distribution, cell migration and apoptosis were determined. We also analyzed the functions of zinc finger protein 451 (ZNF451), which shares a gene sequence with two exons of BC032020 and a non­coding region with another two exons, in PDAC cells. The AsPC­1 and PANC­1 cells that overexpressed BC032020 were used to establish a subcutaneous tumor xenograft model in order to examine the effects of BC032020 on tumor growth in vivo. The results revealed that the BC032020 levels in the PDAC tumor tissues were lower than those in the adjacent normal tissues, and ZNF451 expression inversely correlated with the BC032020 levels in the PDAC tumor tissues and cell lines. BC032020 overexpression led to a decrease in ZNF451 expression; it also suppressed the proliferation and migration of the AsPC­1 and PANC­1 cells, and induced G1 phase arrest and cell apoptosis. The results of in vivo experiments revealed that BC032020 suppressed tumor growth in a xenograft model by inhibiting ZNF451 expression. Taken together, the findings of this study indicate that BC032020 suppresses the survival of PDAC cells by inhibiting ZNF451 expression.


Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/biossíntese , Fatores de Transcrição/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Bases de Dados Genéticas , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
5.
Cell Physiol Biochem ; 43(5): 2062-2073, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29232661

RESUMO

BACKGROUND: Evidence shows that long non-coding RNAs (lncRNAs) are involved in individual development, cell differentiation, cell cycle processes and other important life processes and are closely related to major human diseases, including diabetes. Recent studies have reported that lncRNAs are involved in ß cell functions and that lncRNA Gas5 levels decreased in T2DM patients' serum. The purpose of this study was to clarify the role of lncRNA Gas5 in mouse ß cell functions in vitro and in vivo. METHODS: lncRNA Gas5 expression in T2DM and normal mouse tissues was analyzed using qRT-PCR. RNAi, qRT-PCR, Annexin V-FITC assays, western blot, GSIS and RIA were performed to detect the effects of lncRNA Gas5 on insulin synthesis and secretion in vitro and in vivo. RESULTS: The lncRNA Gas5 level was significantly decreased in db/db mice. However, lncRNA Gas5 expression was relatively high in the pancreas of normal mice. Knockdown of lncRNA Gas5 expression led to cell cycle G1 arrest and impaired insulin synthesis and secretion in Min6 cells. Further, knockdown of lncRNA Gas5 expression in primary isolated islets resulted in decreased expression of insulin gene and transcription factors, Pdx1 and MafA. These results indicate that lncRNA Gas5 might perform as a new regulator, maintaining ß cell identity and function by affecting insulin synthesis and secretion.


Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética
6.
J Biol Chem ; 292(52): 21264-21281, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29109143

RESUMO

The tumor microenvironment is characterized by nutrient-deprived conditions in which the cancer cells have to adapt for survival. Serum starvation resembles the growth factor deprivation characteristic of the poorly vascularized tumor microenvironment and has aided in the discovery of key growth regulatory genes and microRNAs (miRNAs) that have a role in the oncogenic transformation. We report here that miR-874 down-regulates the major G1/S phase cyclin, cyclin E1 (CCNE1), during serum starvation. Because the adaptation of cancer cells to the tumor microenvironment is vital for subsequent oncogenesis, we tested for miR-874 and CCNE1 interdependence in osteosarcoma cells. We observed that miR-874 inhibits CCNE1 expression in primary osteoblasts, but in aggressive osteosarcomas, miR-874 is down-regulated, leading to elevated CCNE1 expression and appearance of cancer-associated phenotypes. We established that loss of miR-874-mediated control of cyclin E1 is a general feature of osteosarcomas. The down-regulation of CCNE1 by miR-874 is independent of E2F transcription factors. Restoration of miR-874 expression impeded S phase progression, suppressing aggressive growth phenotypes, such as cell invasion, migration, and xenograft tumors, in nude mice. In summary, we report that miR-874 inhibits CCNE1 expression during growth factor deprivation and that miR-874 down-regulation in osteosarcomas leads to CCNE1 up-regulation and more aggressive growth phenotypes.


Assuntos
Ciclina E/fisiologia , MicroRNAs/fisiologia , Proteínas Oncogênicas/fisiologia , Osteossarcoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Ciclina E/genética , Ciclina G1/metabolismo , Regulação para Baixo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Oncogênicas/genética , Oncogenes , Osteossarcoma/genética , Fase S
7.
Proc Natl Acad Sci U S A ; 114(38): E8007-E8016, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28874574

RESUMO

The inhibitor NU 2058 [6-(cyclohexylmethoxy)-9H-purin-2-amine] leads to G1-phase cell cycle arrest in the marine diatom, Phaeodactylum tricornutum, by binding to two cyclin-dependent kinases, CDKA1 and CDKA2. NU 2058 has no effect on photosynthetic attributes, such as Fv/Fm, chlorophyll a/cell, levels of D2 PSII subunits, or RbcL; however, cell cycle arrest leads to unbalanced growth whereby photosynthetic products that can no longer be used for cell division are redirected toward carbohydrates and triacylglycerols (TAGs). Arrested cells up-regulate most genes involved in fatty acid synthesis, including acetyl-CoA carboxylase, and three out of five putative type II diglyceride acyltransferases (DGATs), the enzymes that catalyze TAG production. Correlation of transcriptomes in arrested cells with a flux balance model for P. tricornutum predicts that reactions in the mitochondrion that supply glycerate may support TAG synthesis. Our results reveal that sources of intermediate metabolites and macromolecular sinks are tightly coupled to the cell cycle in a marine diatom, and that arresting cells in the G1 phase leads to remodeling of intermediate metabolism and unbalanced growth.


Assuntos
Organismos Aquáticos/metabolismo , Diatomáceas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Mitocôndrias/metabolismo , Transcriptoma/fisiologia , Organismos Aquáticos/genética , Diatomáceas/genética , Mitocôndrias/genética
8.
Biomed Pharmacother ; 92: 661-671, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28586740

RESUMO

Umbelliferone (UMB) has widespread pharmacological activity, comprising anti-inflammatory, anti-oxidant, anti-genotoxic and anti-immunomodulatory but the anticancer activity remains unknown in human oral carcinoma (HOC) KB cells. MTT assay determinations was revealed that treatment of KB cells with UMB, prevent and reduce the cell proliferation with the IC50 - 200µM as well as induces loss of cell viability, morphology change and internucleosomal DNA fragmentation in a concentration dependent manner. Acridine orange and ethidium bromide dual staining assay established that UMB induced apoptosis in KB cells in a dose dependent manner. Alkaline comet assay determination revealed UMB has the potential to increase oxidative DNA damage in KB cells through DNA tail formation significantly (p<0.05). Furthermore, UMB brought a dose-dependent elevation of reactive oxygen species (ROS), which is evidenced by the DCF fluorescence, altered the mitochondrial membrane potential in KB cells. Similarly, we observed increased DNA damage stimulated apoptotic morphological changes in UMB treated cells. Taken together, the present study suggests that UMB exhibits anticancer effect on KB cell line with the increased generation of intracellular ROS, triggered oxidative stress mediated depolarization of mitochondria, which contributes cell death via DNA damage as well as cell cycle arrest at G0/G1 phase. The results have also provided us insight in the pharmacological backgrounds for the potential use of UMB, to target divergent pathways of cell survival and cell death. To conclude UMB could develop as a novel candidate for cancer chemoprevention and therapy, which is our future focus and to develop a connectivity map between in vivo and in vitro activity.


Assuntos
Apoptose/fisiologia , Dano ao DNA/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Neoplasias Bucais/metabolismo , Estresse Oxidativo/fisiologia , Umbeliferonas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos
9.
Mol Biol Cell ; 28(15): 2123-2134, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28539406

RESUMO

The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin-CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/CCdh1 continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.


Assuntos
Centríolos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Quinases Relacionadas a NIMA/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Centríolos/fisiologia , Cílios , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Replicação do DNA , Fase G1/fisiologia , Humanos , Fosforilação , Fase S/fisiologia
10.
FASEB J ; 31(7): 2925-2936, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28360195

RESUMO

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCFßTrCP/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCFßTrCP/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Plasmídeos , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Contendo Repetições de beta-Transducina/genética
11.
Oncogene ; 36(32): 4551-4561, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28368401

RESUMO

Circular RNAs (circRNAs) represent a class of non-coding RNAs that are widely expressed in mammals. However, it is largely unknown about the function of human circRNAs and the roles of circRNAs in human oral squamous cell carcinomas (OSCC). Here we performed a comprehensive study of circRNAs in human OSCC using circRNA and mRNA microarrays, and identified many circRNAs that are differentially expressed between OSCC tissue and paired non-cancerous matched tissue. We further found a circRNA termed circRNA_100290 that served as a critical regulator in OSCC development. We discovered that circRNA_100290 was upregulated and co-expressed with CDK6 in OSCC tissue. Knockdown of circRNA_100290 decreased expression of CDK6 and inhibited proliferation of OSCC cell lines in vitro and in vivo. Via luciferase reporter assays, circRNA_100290 was observed to directly bind to miR-29 family members. Further EGFP/RFP reporter assays showed that CDK6 was the direct target of miR-29b. Taken together, we conclude that circRNA_100290 may function as a competing endogenous RNA to regulate CDK6 expression through sponging up miR-29b family members. Taken together, it indicates that circRNAs may exert regulatory functions in OSCC and may be a potential target for OSCC therapy.


Assuntos
Carcinoma de Células Escamosas/metabolismo , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , RNA/metabolismo , Animais , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Quinase 6 Dependente de Ciclina/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Perfilação da Expressão Gênica , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Regulação para Cima
12.
Tumour Biol ; 39(3): 1010428317694309, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28347242

RESUMO

Lung cancer, of which non-small cell lung cancer accounts for 80%, remains a leading cause of cancer-related mortality and morbidity worldwide. Our study revealed that the expression of WD repeat containing antisense to P53 (WRAP53) is higher in lung-adenocarcinoma specimens than in specimens from adjacent non-tumor tissues. The prevalence of WRAP53 overexpression was significantly higher in patients with tumor larger than 3.0 cm than in patients with tumor smaller than 3.0 cm. The depletion of WRAP53 inhibits the proliferation of lung-adenocarcinoma A549 and SPC-A-1 cells via G1/S cell-cycle arrest. Several proteins interacting with WRAP53 were identified through co-immunoprecipitation and liquid chromatography/mass spectrometry. These key proteins indicated previously undiscovered functions of WRAP53. These observations strongly suggested that WRAP53 should be considered a promising target in the prevention or treatment of lung adenocarcinoma.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Carcinogênese/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Telomerase/biossíntese , Células A549 , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Biologia Computacional , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Fase S/fisiologia , Telomerase/genética
13.
Nucleic Acids Res ; 45(3): 1233-1254, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28180289

RESUMO

Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase ε 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , DNA Polimerase II/química , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/citologia , Olho/crescimento & desenvolvimento , Olho/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genoma de Inseto , Masculino , Microscopia Eletrônica de Varredura , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Neurochem Res ; 42(4): 997-1005, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27995497

RESUMO

Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.


Assuntos
Dano ao DNA/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glioma/metabolismo , Piranos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dano ao DNA/fisiologia , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Piranos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
15.
FASEB J ; 31(1): 132-147, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27694478

RESUMO

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.-Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.


Assuntos
Endonucleases Flap/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Coração/crescimento & desenvolvimento , Oxigênio , Sequência de Aminoácidos , Animais , Dano ao DNA , Feminino , Fibroblastos , Endonucleases Flap/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Coração/embriologia , Masculino , Camundongos , Estresse Oxidativo , Fosforilação , Mutação Puntual
16.
Exp Cell Res ; 350(1): 140-146, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27884681

RESUMO

BACKGROUND: Low extracellular pH (pHe) is a common hallmark of tumor microenvironment, which will also affect pH sensitive T-lymphocytes in this environment. Due to the growing interest on T-cell mediated cancer therapies, acidic stress induced consequences on this lymphocyte deserves through investigations. RESULTS: In line with our previous study [Kim et al., Biochem. Biophys. Res. Commun. 2016; 472(4): 585-91.], we applied sub-lethal acidic stress (pH 3.3, 37°C for 25min) to Jurkat T-lymphocytes. Progression from early apoptosis into late apoptosis was clearly observed by flow cytometry within 3 days. Treatment led to onset of G1 arrest in the first 24h and cell cycling data corresponded to survival of an invasive alkaline phosphatase (AP) positive population. Concerning the massive cell death observed after 72h, both mRNA level (qRT-PCR) and protein level (western blotting) data indicate programmed cell death through p53-p21 independent signaling. CONCLUSION: Taken together, the results obtained suggest that the majority of Jurkat cells exposed to short but intense acidic stress conditions, as used here, undergo intrinsic apoptosis, while invasion and AP activation only occurred in a small surviving cell population.


Assuntos
Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Divisão Celular , Citometria de Fluxo/métodos , Humanos , Concentração de Íons de Hidrogênio , Células Jurkat , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo
17.
Cancer Biother Radiopharm ; 31(7): 261-7, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27610469

RESUMO

Deregulated expressions of mucins have been found in various malignancies and play a pivotal role in carcinogenesis. MUC5AC, as a secreted mucin, is reported to be aberrantly expressed during epithelial cancer progression, including colon cancer. However, the mechanisms of the oncoprotein MUC5AC in the initiation of colon cancer requires further investigation. Here, we collected colon cancer tissues (n = 20) and corresponding paracancerous tissues (n = 20) and found that the expression of MUC5AC was significantly elevated in colon cancer tissues when compared with the corresponding paracancerous tissues. Immunofluorescence indicated that all colon cancer cell lines, including HT29, SW620, and the normal human intestinal epithelial cells FHC, showed the positive expression of MUC5AC, and SW620 exhibited the highest expression. Moreover, knockdown of MUC5AC in SW620 cells remarkably suppressed cell vitality and promoted apoptosis and G1 cell cycle arrest, resulting in the impaired ability of colony formation. Furthermore, the inhibition of MUC5AC in SW620 cells dramatically repressed the cell migration and invasion. These results demonstrated that MUC5AC as an oncogene could be a promising target in the treatment of colon cancer.


Assuntos
Neoplasias do Colo/metabolismo , Mucina-5AC/biossíntese , Apoptose/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HT29 , Humanos , Mucina-5AC/deficiência , Mucina-5AC/genética , Mucina-5AC/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção
18.
Sci Rep ; 6: 32172, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27572135

RESUMO

Data derived from genomic and transcriptomic analyses have revealed that long noncoding RNAs (lncRNAs) have important roles in the transcriptional regulation of various genes. Recent studies have identified the mechanism underlying this function. To date, a variety of noncoding transcripts have been reported to function in conjunction with epigenetic regulator proteins. In this study, we investigated the function of linc00598, which is transcribed by a genomic sequence on chromosome 13, downstream of FoxO1 and upstream of COG6. Microarray analysis showed that linc00598 regulates the transcription of specific target genes, including those for cell cycle regulators. We discovered that linc00598 regulates CCND2 transcription through modulation of the transcriptional regulatory effect of FoxO1 on the CCND2 promoter. Moreover, we observed that knockdown of linc00598 induced G0/G1 cell cycle arrest and inhibited proliferation. These data indicate that linc00598 plays an important role in cell cycle regulation and proliferation through its ability to regulate the transcription of CCND2.


Assuntos
Ciclina D2/biossíntese , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , RNA Longo não Codificante/metabolismo , Elementos de Resposta/fisiologia , Transcrição Genética/fisiologia , Ciclina D2/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Humanos , Células K562 , Células MCF-7 , RNA Longo não Codificante/genética , Fase de Repouso do Ciclo Celular/fisiologia , Células THP-1
19.
Tumour Biol ; 37(9): 12823-12831, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27449035

RESUMO

Cullin1 (Cul1) is a scaffold protein of the ubiquitin E3 ligase Skp1/Cullin1/Rbx1/F-box protein complex, which ubiquitinates a broad range of proteins involved in cell-cycle progression, signal transduction, and transcription. To investigate the role of Cul1 in the development of renal cell carcinoma (RCC), we evaluated the Cul1 expression by immunohistochemistry using a tissue microarray (TMA) containing 307 cases of RCC tissues and 34 normal renal tissues. The Cul1 expression was increased significantly in RCC and was correlated with renal carcinoma histology grade (P = 0.007), tumor size (P = 0.013), and pT status (P = 0.023). Also, we found that silencing of Cul1 leads to increased expression of p21 and p27 that could inhibit the cyclin D1 and cyclin E2 expressions and arrest cell cycle at the G1 phase. Furthermore, knockdown of Cul1 inhibits RCC cell migration and invasion abilities by up-regulating the expression of TIMP-1 which could inhibit the expression of MMP-9. Finally, using bioluminescence imaging, we found that Cul1 knockdown significantly reduced the tumor growth in vivo. Cul1 may constitute a potential therapeutic target in RCC.


Assuntos
Carcinoma de Células Renais/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas Culina/biossíntese , Neoplasias Renais/metabolismo , Animais , Western Blotting , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas Culina/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Neoplasias Renais/terapia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Interferência de RNA , Terapêutica com RNAi/métodos , Análise Serial de Tecidos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
20.
Am J Physiol Cell Physiol ; 311(2): C179-89, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27170637

RESUMO

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3-14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2'-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27(kip1)), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis.


Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Fase G1/fisiologia , Proteínas de Homeodomínio/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Animais , Apoptose/fisiologia , Artérias Carótidas/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
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